Laser shock processing (LSP) is an effective and promising technology for improving surface mechanical properties of metals. The study of the strain behavior of individual phase of advanced engineering materials with polycrystalline and dual-phase microstructures subjected to laser shock processing is an important emerging frontier, which facilitates understanding of the relative roles of intrinsic and extrinsic attributes of microstructure upon strengthening; compared with the strengthening process of metals at the macroscopic scale of deformation. The influence of LSP on the surface layer properties and microstructures of a Ti-6Al-4V alloy has been investigated focusing on the microstructure response of the surface layer of the alloy by means of high efficient Nd3+ : YAG ceramic pulse laser with 12.5 J per pulse at 1064 nm and 10 Hz repetition rate. The microstructures response of the alloy are analyzed and characterized with by FE-SEM; TEM and the inverse fast fourier transform (IFFT) algorithm; respectively. The experimental results show that the surface hardness of the laser shocked Ti-6Al-4V alloy can increase 80%; and the compressive residual stress can be over 500 MPa. Obvious preference effect between alpha and beta phase is discovered upon strengthening of the alloy under the conditions of the ultra high energy and ultra-high strain rate of laser shock. With the lower shock energy, the deformation strengthening of beta phase takes precedence over the other; as the shock energy increasing, both alpha and beta are strengthened simultaneously, whereas, the previously strengthened beta phase shows saturated strengthening effect. The results also reveal that dislocation multiplication is the main strengthen mechanism in the laser shocked region, including oriented dislocation projection and dislocation dipoles in the alpha phase with hcp crystal lattice, but diversified configurations, such as edge-dislocation, extended dislocations and dislocation dipoles presenting in the beta phase with bcc crystal lattice. The semi-coherent interface with misfit dislocations between alpha and beta phase boundary is discovered, which plays a synergetic role upon deformation strengthening. Additionally, the strain screening manifestation within the laser shocked region is also discussed, which is regarded as a kind of self-organization phenomenon of deformation defects, and can be attributed to the synthetic effect of the confinement conditions upon laser shocking, the accumulative strengthening mode of single-spot laser shocking process and the differences of strength and crystalline structure between the lamellar alpha and beta phases.